Epidermal growth factor (EGF) is a 6.2 kDa polypeptide that specifically binds to the epidermal growth factor receptor (EGFR). EGF contains 53 amino acids with three internal disulfide bridges, and has the amino acid sequence shown in SEQ ID NO:1.
Binding of EGF to its receptor induces a conformational change in the receptor and receptor aggregation (Greenfield, et al., EMBO J. 8:4115-4123, 1989; Varden and Schlessinger, Biochem, 26: 1443-1451, 1987). Receptor aggregation stimulates an intrinsic tyrosine kinase activity in the cytoplasmic domain of EGFR, which in turn leads to recruitment and phosphorylation of other substrates, resulting in mitogenic signaling and/or a variety of other cellular activities (Paw on and Schlessinger, Curr. Biol. 3:434-442, 1994; Alroy and Varden, FEBS Lett. 410:83-86, 1997; Riese and Stern, Bioessays 20:41-48, 1998).
Therapies that promote EGFR signaling find use in treating a diverse range of conditions. EGF super-agonists have long been sought due to their potential applications in wound healing, tissue engineering and regenerative medicine. For example, stimulation of EGFR with EGF has been shown to accelerate wound healing, (e.g., in gastric and oral ulcers, diabetic foot ulcers, skin grafts, corneal epithelial wounds, and tympanic membrane perforations (Milani and Calabro, Microsc. Res. Tech. 52:360-371, 2001; Fujisawa, et al, J. Oral Pathol. Med. 32:358˜366, 2003; Bennett, et al, Br J Surg. 90:133˜146, 2003: Brown, et al, N. Engl. J. Med. 321:7˜79, 1989; Lu, at al, Exp. Biol. Med. (Maywood) 226:653˜64, 2001; Ma, at al, Acta Otolaryngol. 122:586˜599, 2002). As another example, stimulation of EGFR has been demonstrated to regulate nerve regeneration and atherogenesis (Xian and Zhou, Mol Neurobiol 20: 157-183, 1999; Lamb, et al, Atherosclerosis 168: 191-194, 2003).
Historically, attempts to discover EGF agonists with improved biological activity by means of screening for EGFR kinase activity, reporter gene expression, or increased binding affinity to EGFR have met with limited success (see, e.g., Coco et al., (2002). Nat Biotechnol 20, 124-50; Sounau et al., (1997) Nucleic Acids Res 25, 1585-90: Souriau et al., Biol Chem 380). Similarly, attempts to engineer such mutants have faded to yield molecules with the desired activity (see e.g. U.S. Pat. No. 5,547,935, issued to Mullenbach et al. and U.S. Pat. No. 7,084,246, issued to Coco et al., hereby incorporated by reference in their entirety), due to a previous lack of understanding as to aspects of the process by which receptor binding effects cellular changes, i.e., as to intracellular trafficking and downstream signaling.
There is therefore a long-felt and previously unmet need in the art for EGF polypeptides with improved biological activity as compared to the activity exhibited by the wild-type molecule. Further, there is a long-felt and previously unmet need in the art for a high-throughput method of identifying mutant EGF polypeptides having improved biological activity as compared to the activity of wild-type EGF, which method is both effective and not excessively costly in terms of money, time and effort. Further, there is a long-felt and previously unmet need in the art for a method of rationally engineering mutant EGF polypeptides having improved biological activity. Further, there is a long-felt and previously unmet need in the art for an effective method of treating wounds using mutant EGF polypeptides having improved biological activity.
The present invention addresses these issues.